Claims:

1. A method for transcriptional control of an endogenous gene,
comprising:introducing, into a suitable region of a target endogenous
mammalian target gene sequence of a mammalian cell having a genome, at
least one cis element of at least one exogenous binary regulatory DNA
sequence;introducing, into the genome of the mammalian cell, an
expression cassette/vector encoding at least one corresponding trans
element of the at least one binary regulatory DNA sequence, wherein the
at least one cis element is operative with the target gene sequence and
the at least one trans element to provide for transcriptional control of
the endogenous target gene expression by the at least one binary
regulatory DNA sequence.

2. The method of claim 1, wherein introducing of the at least one cis
element comprises introducing multiple cis elements.

3. The method of claim 1, wherein introducing of the at least one trans
element comprises introducing multiple trans elements.

13. The method of any one of claims 1 through 12, comprising:introducing
the at least one cis element into at least one cell of a first transgenic
mammal;introducing the at least one trans element into at least one cell
of a second transgenic mammalcrossing the first and second transgenic
mammals to provide at least one cell, or provide at least one progeny
animal with at least one cell, wherein the at least one cis element is
operative with the target gene and the trans element to provide for
transcriptional control of the endogenous gene expression in the at least
one cell by the at least one binary regulatory DNA sequence.

14. A mammalian cell, comprising at least one of:an endogenous target gene
sequence having suitably inserted therein at least one cis element of at
least one exogenous binary regulatory DNA sequence; andan expression
cassette/vector, within the genome of the mammalian cell, encoding at
least one corresponding trans element of the at least one binary
regulatory DNA sequence, wherein the at least one cis element is
operative with the target gene sequence and the at least one trans
element to provide for transcriptional control of the endogenous target
gene expression by the at least one binary regulatory DNA sequence.

15. The cell of claim 14, wherein the cell is that of, or within a
transgenic animal.

20. The lacI repressor of claim 19, wherein the lacI sequence comprises a
consensus GCCACCATGG (SEQ ID NO:1), or GNCACCATGG (SEQ ID NO:2) sequence,
wherein the N is selected from the group consisting of cytosine, guanine,
and thymidine.

21. A lacI repressor, comprising at least one mutation selected from the
group consisting of proline to tyrosine change at the third amino acid
residue and serine to leucine change at amino acid residue 61.

22. The lacI repressor, of any claims 18 through 21, wherein the lacI
repressor is further fused to another protein, wherein the another
protein is VP16.

25. A method for bidirectional cloning, comprising:generating at least one
deletion mutant construct; andinserting at least one regulatory sequence
at different locations with respect to the at least one deletion mutant
construct, wherein only three restriction enzymes are used, and wherein
the generating at least one deletion mutant construct and the inserting
at least one regulatory sequence are synchronized.

26. The method of claim 21, wherein the regulatory sequence is an
operator.

27. The method of claim 22, wherein the operator is selected from the
group consisting of the lac operator, the lac operator with at least one
lacI binding consensus sequence, the lac operator with at least one lacI
binding symmetrical sequence TGTGGAATTGTGAGC-GCTCACAATTCCACA (SEQ ID
NO:3), the tet operator, and the Gal4 regulatory sequence.

28. The method of claim 1, further comprising modifying the number or
pattern of CpG dinucleotides within the expression cassette/vector to
modulate DNA methylation-mediated silencing thereof.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application claims the benefit of priority to U.S. Provisional
Patent Application Ser. No. 61/226,234 filed Jul. 16, 2009 and entitled
CONTROL OF ENDOGENOUS DNMT1 GENE EXPRESSION BY EXOGENOUS BINARY
REGULATORY SYSTEMS, which is incorporated by reference herein in its
entirety.

FIELD OF THE INVENTION

[0003]Aspects of the present invention relate generally to regulation of
gene expression, and more particularly to novel and efficacious
compositions and methods for control of endogenous gene expression,
including but not limited to transcriptional control of endogenous genes
in mammalian systems using cis/trans binary regulatory DNA sequences.

BACKGROUND

[0004]Several techniques have been developed to probe gene function
through either loss or gain of function analysis in mice, such as
conventional/conditional knock-outs and transgenics. However, cell
lethality has limited the successful implementation of knockout
technologies for some genes in, e.g., cells and transgenics. In vivo
knockdown approaches relying on tet-regulated SiRNA expression may be
able to address this problem for some genes, but SiRNA development has
proved challenging for some genes, and the degree of repression by RNA
interference varies.

[0005]In mammals, DNA methylation is a covalent enzymatic modification
occurring at C-5 position of cytosine ring in the context of CpG
dinucleotide, cytosine followed by guanine. This reaction is performed by
the enzyme called DNA methylatransferase. There exist at least three
known functional DNA methyltransferases in mammal, Dnmt1, Dnmt3a, and
Dnmt3b (1). Dnmt1 is responsible for maintaining methylation content upon
DNA replication based on the methylation information on the parent
strand, so it is called maintenance methylatransferase. Dnmt3a and Dnmt3b
are called de novo methyltransferase since they can function
independently of methylation status of the parent strand (2). CpG
dinucleotide is severely underrepresented in most part of mammalian
genome due to the spontaneous deamination of methylated cytosine,
resulting in C to T transition. However, there are regions called CpG
islands, which are rich in CpG content (3). CpG islands are often
associated with promoters of genes. About a half of the promoters of the
genes in mammal have a CpG island (3). In general, CpG islands are not
methylated in normal cells with a few exceptions, such as imprinted genes
and genes on inactive X chromosome (2). In cancer cells, however, some
CpG islands become methylated, resulting in abnormal gene silencing.
Hypermethylated CpG islands are often associated with tumor suppressor
genes, suggesting the important role of DNA methylation in tumorigenesis
(4). Surprisingly, the silencing of classic tumor suppressor genes by
aberrant promoter hypermethylation is at least as common as the
disruption of classic tumour suppressor genes by genetic mutation in
human cancer (5). However, the mechanisms in which this abnormal promoter
hypermethylation occurs still remain largely unknown.

[0006]There is, therefore a pronounced need in the art for novel and
efficacious compositions and methods for transcriptional control of
endogenous genes (e.g., in a mammalian cell or cell system), including
but not limited to efficacious methods for control of endogenous gene
expression of the major DNA methyltransferase genes (e.g., Dnmt1).

SUMMARY OF THE INVENTION

[0007]Particular aspects provide novel and efficacious methods for control
of endogenous gene expression. The inventive methods represent the first
demonstration of transcriptional control of an endogenous gene in a
mammalian system. In certain aspects, this can be applied to any gene and
serve as a useful tool, particularly where conventional conditional
knock-out and transgenic approaches cause cell lethality, and/or where
SiRNA approaches have failed.

[0008]Certain aspects provide efficacious methods for control of
endogenous gene expression of the major DNA methyltransferase, Dnmt1
gene, and provide fine-tuned temporo-spatial control of endogenous Dnmt1
gene expression to enable, for example, elucidation and modulation of DNA
methylation and other epigenetic mechanisms in cancer.

[0009]In particular embodiments, the novel methods comprise a novel
transcriptional regulatory system involving three exogenous binary
systems, lacI, tetR and Gal4, which allow for the reversible up- and
down-regulation of a target gene from its endogenous locus.

[0010]In particular aspects, a number of modifications were engineered
into both the lac operator and repressor, which significantly improved
the repression as compared to wild type (WT) lac and tet systems.

[0011]In certain embodiments, lac operator sequences were introduced into
the endogenous Dnmt1 promoter through gene-targeting, without
significantly altering promoter activity in the absence of repressors.
Real-time RT-PCR analysis of targeted ES cells showed that the expression
of the targeted allele was substantially reduced by the introduction of
lac repressor to less than 10% of WT allele. In particular embodiments,
the lacO targeted Dnmt1 allele has been introduced into the mouse
germline, to provide a respective transcriptional control system in vivo.

[0012]In additional embodiments, two binary systems, tet operator/tetVP16
and Gal4 binding sequence/Gal4VP16 were introduced to achieve
upregulation of Dnmt1. In particular aspects, luciferase reporter assays
are used in assays to modulate expression from the Dnmt1 promoter over
two orders of magnitude, ranging from 3.7% to 800% of unregulated
expression by combining these three binary systems.

[0013]In further aspects, gene targeting experiments are conducted in
mouse ES cells with a Dnmt1 promoter targeting construct that combines
the cis elements for all three of these systems.

[0014]In certain embodiments, endogenous Dnmt1 expression was successfully
up and down regulated in a reversible temporal-spatial manner in mice. In
further embodiments, this technology was potent enough to reproduce the
embryonic lethal phenotype of genetic knock-out and to attenuate
transcription elongation, and the lethal phenotype was rescued by IPTG
treatment. This establishes the first paradigm of experimental rescue of
an embryonic lethal phenotype in loss-of-function genetics, and enables
study of regulated expression for genes for which in vivo
characterization have been limited in the prior art due to a lethal
phenotype.

[0015]Certain aspects provide for controlling eukaryotic (e.g., mammalian)
gene expression through the institution of a physical access to the
endogenous promoter, and in exemplary embodiments with Dnmt1, a gene for
which the use of conventional/conditional knock-outs and transgenic
approaches have been limited due to embryonic or cell lethality. This
invention, however, is not limited to the Dnmt1 gene, and rather is
generally applicable to other genes, other organisms. and other systems.

[0016]According to particular embodiments, the current invention relates
to a method for transcriptional control of an endogenous gene,
comprising: introducing, into a suitable region of a target endogenous
mammalian target gene sequence of a mammalian cell having a genome, at
least one cis element of at least one exogenous binary regulatory DNA
sequence; introducing, into the genome of the mammalian cell, an
expression cassette/vector encoding at least one corresponding trans
element of the at least one binary regulatory DNA sequence, wherein the
at least one cis element is operative with the target gene sequence and
the at least one trans element to provide for transcriptional control of
the endogenous target gene expression by the at least one binary
regulatory DNA sequence. According to additional embodiments, the
transcriptional control involves introducing multiple cis elements.
According to further embodiments, introducing of the at least one trans
element comprises introducing multiple trans elements. According to still
further embodiments, the expression cassette/vector encoding the at least
one corresponding trans element of the binary regulatory DNA sequence
comprises mammalian promoter and/or regulatory sequences.

[0017]In certain aspects, the transcriptional control comprises
administration of an agent suitable to modulate the intracellular
interaction between the cis and trans elements of the binary regulatory
DNA sequences. In further aspects, the agent is selected from the group
consisting of allolactose, lactose, IPTG, tetracyclines, and galactose.
In still further aspects, the cis and trans exogenous binary regulatory
DNA sequences are of heterologous origin. In yet further aspects, the cis
and trans exogenous binary regulatory DNA sequences are from
microorganisms, including bacteria and yeast.

[0018]According to certain aspects, the binary regulatory DNA sequences
are selected from the group consisting of lac operator/repressor, tet
operator/repressor, Gal4 operator/repressor, and functional variants
(muteins, fusions, deletions, insertions, fragments, derivatives, etc)
thereof. According to further aspects, transcriptional control of the
endogenous gene expression by the binary regulatory DNA sequences
comprises transcriptional repression or transcriptional activation.
According to yet further aspects, introducing at least one cis element of
at least one exogenous binary regulatory DNA sequence comprises
recombination. In particular aspects, the target gene comprises the Dnmt1
gene. In further aspects the method of the invention comprises:
introducing the at least one cis element into at least one cell of a
first transgenic mammal; introducing the at least one trans element into
at least one cell of a second transgenic mammal crossing the first and
second transgenic mammals to provide at least one cell, or provide at
least one progeny animal with at least one cell, wherein the at least one
cis element is operative with the target gene and the trans element to
provide for transcriptional control of the endogenous gene expression in
the at least one cell by the at least one binary regulatory DNA sequence.

[0019]According to particular embodiments, the current invention includes
a mammalian cell, comprising at least one of: an endogenous target gene
sequence having suitably inserted therein at least one cis element of at
least one exogenous binary regulatory DNA sequence; and an expression
cassette/vector, within the genome of the mammalian cell, encoding at
least one corresponding trans element of the at least one binary
regulatory DNA sequence, wherein the at least one cis element is
operative with the target gene sequence and the at least one trans
element to provide for transcriptional control of the endogenous target
gene expression by the at least one binary regulatory DNA sequence.
According to further aspects, the cell is that of, or within a transgenic
animal. According to yet further aspects, the cell is an embryonic stem
cell. According to still further aspects, the target gene comprises the
Dnmt1 gene.

[0020]In certain embodiments, a lacI repressor comprises a stabilizing
amino acid adjacent to the N-terminal lysine. In further exemplary
embodiments, the stabilizing amino acid is, for example, Gly, Ala, or
Val. In yet further embodiments, the lacI sequence comprises a consensus
GCCACCATGG (SEQ ID NO:1) sequence (or GNCACCATGG (SEQ ID NO:2) sequence,
wherein the N is selected from the group consisting of cytosine, guanine,
and thymidine.), or comprises the above described stabilizing amino acid
in combination with comprising said consensus sequence.

[0021]According to particular embodiments, a lacI repressor, comprises at
least one mutation selected from the group consisting of proline to
tyrosine change at the third amino acid residue and serine to leucine
change at the 61st amino acid residue. According to further
embodiments, the lacI repressor (or variant, or biologically active
portion thereof) is fused to another protein, wherein the other protein
is, for example, VP16. Particular embodiments provide a lacI repressor,
comprising a fusion protein, wherein the fusion protein is VP16.
Particular embodiments provide a lac operator, comprising a symmetric lac
operator sequence consisting, for example, of
TGTGGAATTGTGAGC-GCTCACAATTCCACA (SEQ ID NO:3).

[0022]In certain aspects, the invention involves a method for
bidirectional cloning, comprising: generating at least one deletion
mutant construct; and inserting at least one binary regulatory DNA
sequence at different locations with respect to the at least one deletion
mutant construct, wherein only three restriction enzymes (e.g., BsaI,
AvrII and SpeI are used, and wherein the generating at least one deletion
mutant construct and the inserting at least one regulatory sequence are
synchronized. In further aspects, the regulatory sequence is an operator.
In still further aspects, the operator is selected from the group
consisting of the lac operator, the tet operator, and the Gal4 regulatory
sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a schematic view of the binary transcriptional regulatory
system. According to exemplary aspects of the present invention, the this
regulatory system is made by crossing a mouse transgenic line expressing
either a repressor or activator or both driven by a tissue specific
promoter with a mouse line in which the endogenous promoter of a target
gene is modified with operator sequences that are controlled by the
repressor or activator. This system allows for transcriptional
regulation, either up or down expression, upon the binding of activators
or repressors to the operators, respectively.

[0024]FIG. 2 shows, according to exemplary aspects of the present
invention, the first demonstration, using real-time PCR, of repressing an
endogenous gene expression with lac operator/repressor.

[0025]FIG. 3 indicates the comparison of repression capacity between Lac
and Tet repressors. Panel A shows the reporter and repressor constructs
used in the transient report assay; non-functional lacI (NFlacI), wild
type lacI (Lad), lacI with tight binding mutation (LacIY), wild type tet
repressor (TetR), tet repressor with nuclear localization signal
(tetRNLS), lac operator (O), and tet operator (T). Panel B indicates the
activities of the reporter genes in the absence of repressors; CMV-O and
CMV-T is the reporter containing the lacO or the tetO. Panel C shows the
reporter activities in the presence of repressors as indicated.

[0026]FIG. 4 shows the comparison of repression capacity between Lac and
Tet repressors. Panel A shows the reporter and repressor constructs used
in the transient report assay. Panel B indicates the repression of a
promoter construct having a lacO (O) followed by tetO (T). Panel C shows
the repression of a promoter construct having a tetO (T) followed by a
lacO (O).

[0027]FIG. 5 shows, according to exemplary aspects of the present
invention, inducible and reversible up and down endogenous gene
regulation.

[0028]FIG. 6 shows, according to exemplary aspects of the present
invention, the repressing effect of tight binding mutations.

[0029]FIG. 7 shows, according to exemplary aspects of the present
invention, exemplary stabilizing amino acid insertions for Lac repressor
(SEQ ID NOS:5 and 6). Panel A (top) shows the coding sequence for the
particular amino acids that were inserted in between the first codon
(ATG) and the second codon (AAA). Panel A (bottom) shows the reporter
assay using the stabilized Lac repressor. Panel B (top) shows the
experimental scheme for the quantification assay for the repressors and
(bottom) the results from the scheme.

[0030]FIG. 8 shows, according to exemplary aspects of the present
invention, the effect of stabilized lac repressors (SEQ ID NOS:7-9) in a
reporter assay.

[0031]FIG. 9 indicates, according to exemplary aspects of the present
invention, the results of consensus (O), symmetrical (S), or double
consensus (O) operators in a luciferase reporter assay. Panel A shows the
sequence of the consensus (O) (SEQ ID NO:10) and symmetrical operators
(S) (SEQ ID NO:3) used in the reporter assays. Panel B shows the
luciferase value from reports harboring either the consensus (O) or
symmetrical operator (S). Panel C shows the luciferase activity from
reporter constructs harboring either one (O) or two (O) operators.

[0032]FIG. 10 shows, according to exemplary aspects of the present
invention, the reversible control of the modified Dnmt1 promoter using
IPTG. Panel A (top) shows the reporter construct and the repressors that
were transfected into cells and (bottom) the expression level from the
resulting transfection either in the presence or absence of IPTG. Panel B
shows the expression level from the reporter construct using the
indicated repressor construct and the indicated IPTG concentration.

[0034]FIG. 12 shows, according to exemplary aspects of the present
invention, the characterization of the Dnmt1 promoter. Panel A shows
(top) the genomic configuration of Dnmt1 locus, including the oocyte
(10), somatic (1s), and pachytene (1p) exon 1. In addition, FIG. 12,
panel A shows the sequence homology comparison with rat and human Dnmt1
promoter sequences and transcription factor binding site search (TF
search). Panel A (bottom) demonstrates, via reporter assays, the
expression of the Dnmt1 promoter deletion mutants. Panel B shows, by
reporter assays, the expression of 3' and internal deletion mutants of
the Dnmt1 promoter.

[0035]FIG. 13 shows, according to exemplary aspects of the present
invention, the effect of modifying the Dnmt1 promoter with lacOs. Panel A
shows the expression from the Dnmt1 promoter modified with lac operator
without the lac repressor present. Panel B is a schematic of the Dnmt1
promoter with different forms of the lac operator: symmetric operators
(S) and double operators (O).

[0036]FIG. 14 shows, according to exemplary aspects of the present
invention, the effect of modifying other promoters with the lac operator
sequence. Panel A (top) shows the human ubiquitin C promoter (hUbc) and
SV40 promoter as modified with two copies of lac operators and (bottom)
the level of expression from these modified promoters. Panel B (left)
shows the rabbit beta-globin intron as modified with the symmetric
operators. This modified intron was placed between the Dnmt1 promoter and
the luciferase reporter. On the right, the expression levels from the
Dnmt1 promoter-modified intron-luciferase reporter construct is shown.

[0037]FIGS. 15A-G show, according to exemplary aspects of the present
invention, the sequence of the targeting vector for Dnmt1 promoter that
has been modified with lacO, tetO, gal4 binding sequences (SEQ ID NO:11).

[0038]FIG. 16 shows, according to exemplary aspects of the present
invention, the sequence (SEQ ID NO:12) of the modified lacI with the
stabilizing amino acid (GGC), and the tight binding mutation (TAT)
labeled in red and blue, respectively.

[0039]FIG. 17 shows, according to exemplary aspects of the present
invention, a schematic of the bi-directional cloning strategy.

DETAILED DESCRIPTION OF THE INVENTION

[0040]The ability to control gene expression in living organisms through
genetic manipulations has been essential in advancing our understanding
of physiologic and pathologic processes of biology. Genetic tools in
functional genomics can be broadly categorized into two types;
loss-of-function and gain-of-function approaches. Knock-out technology
via gene-targeting (or targeted transgenesis) has been arguably the most
successful application of the first category. Gene-targeting allows for
the inactivation of a target gene by introducing a genetic
modification(s) at a predetermined site(s) in the genome through
homologous recombination that results in a null mutation (Thomas and
Capecchi, 1986). In contrast to gene-targeting, the transgenic approach
(or random transgenesis) enables the integration of foreign DNA into
genomic sites that are not known a priori (Ristevski, 2005). The random
integration is achieved through injection of transgenes into the male
pronucleus of a fertilized egg or transfection of transgenic constructs
into embryonic stem (ES) cells. This random transgenesis, in general, has
been used to study gene function through elevation of gene expression.

[0041]Both targeted and random transgenesis have produced numerous animal
models that provide valuable functional analyses for many genes and
insights into mechanisms of various biological processes as well as
diseases. However, the disadvantage of these approaches is that genetic
alterations caused by these approaches are germline mutations that are
present in all cell types at all time. This has limited the broad
application of these technologies. For example, germline null mutations
often cause a lethal phenotype, which prevents the functional analysis of
the gene of interest in adult animals. Furthermore, universal gene
activation or inactivation may be subject to a pleiotropic effect that
may complicate the interpretation of outcomes. To overcome these
limitations and to achieve a more precise gene expression control,
conditional gene-targeting and inducible transgenics have been developed
(Chien, 1996; Metzger et al., 1995; Glaser et al., 2005; Gossen and
Bujard, 1992).

[0042]The study of the role of Dnmt1 in tumorigenesis has been limited
because of the non-viability of mice lacking functional Dnmt1. One
alternative approach to overcome this problem is to provide a
conditionally inactivated allele of Dmnt1, in which Dnmt1 will be
inactivated in time and/or tissue specific manner.

[0043]The most commonly used system to generate conditionally inactivated
allele is the Cre-loxP system, in which Cre-mediated deletion of a target
gene occurs in tissue specific manner depending upon the promoters
driving Cre recombinase expression (6). A conditionally inactivated
allele of the mouse Dnmt1 has been successfully generated using Cre-loxP
system (7, 8). However, this system turned out to be unsuitable for the
study of the role of Dnmt1 in early stages of cancer development in vivo
with two reasons. First, differentiated cells that lose functional Dnmt1
expression undergo p53-dependent cell death (7). Second, Cre-based
recombination is a highly stochastic event, that is, in this system some
cells undergo Cre-mediated deletion of Dnmt1 and subject to p53 dependent
cell death, but some other cells do not go through Cre-based deletion of
Dnmt1 and have normal level of Dnmt1 expression. An ideal system to study
the role of DNA methylation in tumorigenesis might be a system in which
all of the targeted cells have partially inactivated Dnmt1 expression
level that do not cause either embryonic lethality or p53-dependant cell
death, instead of a full inactivation. To accomplish this, particular
aspects provide an in vivo binary transcriptional repression system based
on a prokaryotic gene switch system, lac operon similar to the widely
used binary transcriptional activation systems, tet operon (9, 10, 11).
According to particular aspects, the Lac operator/repressor system has a
couple of features that make it better suited for the present purposes as
compared to other prokaryotic operator/repressor systems. First, lac
repressor works as a tetramer, and this tetramer can bind to two lac
operators on one DNA stretch and induce the intervening DNA to form a
loop (12). The distance between two operators can be more than 500 bp.
This feature assists to insert lac operator sequences into Dnmt1 promoter
at locations that do not interfere with promoter function in the absence
of the repressor, yet result in good repression in the presence of the
repressor. According to additional aspects, with other prokaryotic
operator/repressor systems whose repression mechanism relies only on
steric hindrance (13), it may not be possible to insert operator
sequences in eukaryotic promoters at locations that do not impede
promoter function in the absence of the repressor, yet result in good
repression in the presence of the repressor, because eukaryotic promoters
have evolved in the absence of operator/repressor based transcriptional
regulation system, whereas the prokaryotic promoters controlled by the
operon systems have evolved in a way that they can accommodate operator
sequences in vicinity of transcriptionally critical region. Second, the
lac operator/repressor system has the longest operator sequence among
prokaryotic operator/repressor systems, which reduces the probability of
the existence of fortuitous lac operator sequences in the mouse genome.
In particular aspects, the Dnmt1 promoter is modified with lac operators
and transgenic mice are generated that express lac repressor in limited
tissues, using tissue-specific promoters, and in particular aspects of
this system the results are partial inactivation of Dnmt1 expression, not
full inactivation. First, prokaryotic operator/repressor systems have
been shown to be leaky in mammalian cells (14, 15, 16). Second, by
generating several lines of lacI transgenic mice with different copy
numbers and integrated sites, varying degrees of expression levels of lac
repressor, resulting in a range of repression capacities are provided.

[0044]Particular embodiments provide for application of the repression and
activation binary systems to an exemplarly endogenous gene, Dnmt1, such
that cis-elements of the binary systems were integrated into the
endogenous promoter of the target gene, and the repressor and
transactivator induce repression and activation through a direct
interaction with the transcription machinery on the promoter (FIG. 1).

Terms Used Herein

[0045]As used herein, an endogenous gene is one that originated from
within a particular organism, tissue, or cell.

[0046]As used herein, exogenous binary regulatory DNA sequence are
sequences that originated from another gene, or from outside a particular
organism, tissue, or cell and is suitable for controlling the regulation
of a target endogenous gene expression within a particular organism,
tissue, or cell.

[0047]As used herein, an expression cassette/vector is a nucleic acid
sequence that may constitutively express or inducibly express a certain
protein. In certain embodiments, the expression cassette/vector as used
herein encodes for a transcriptional regulator, and is operatively driven
by a promoter that is active in the host cell.

[0048]As used herein, a cis element is a nucleic acid sequence that is
operably linked to the gene of interest and suitable for binding trans
elements, providing for altered transcriptional expression of a certain
target gene operably linked to the cis element.

[0049]As used herein, a trans element is an regulatory element, which can
be DNA, RNA, or protein, suitable for binding to cis elements to alter
transcriptional expression of a certain gene.

[0050]As used herein, DNA or nucleic acid sequences that are referred to
as heterologous origin are those DNA or nucleic acid sequences derived
from an organismal source other than the organism in which it is to be or
has been inserted.

[0051]As used herein, microorganisms, may refer to any microorganism and
includes, but is not limited to bacteria and yeast.

[0052]As used herein, regulatory sequences include but are not limited to
cis elements, such as those examples described herein for illustrative
purposes.

Biologically Active Variants

[0053]Functional variants of the repressors and activators described
herein can be naturally or non-naturally occurring. Naturally occurring
variants (e.g., polymorphisms) are found in humans or other species and
comprise amino acid sequences which are substantially identical to the
amino acid sequences of the repressors and activators as disclosed
herein.

[0054]Non-naturally occurring variants which retain substantially the same
biological activities as naturally occurring protein variants, are also
included here. Preferably, naturally or non-naturally occurring variants
have amino acid sequences which are at least 85%, 90%, or 95% identical
to the amino acid sequences of the repressors and activators as disclosed
herein. More preferably, the molecules are at least 98% or 99% identical.
Percent identity is determined using any method known in the art. A
non-limiting example is the Smith-Waterman homology search algorithm
using an affine gap search with a gap open penalty of 12 and a gap
extension penalty of 1. The Smith-Waterman homology search algorithm is
taught in Smith and Waterman, Adv. Appl. Math. 2:482-489, 1981.

[0055]As used herein, "amino acid residue" refers to an amino acid formed
upon chemical digestion (hydrolysis) of a polypeptide at its peptide
linkages. The amino acid residues described herein are generally in the
"L" isomeric form. Residues in the "D" isomeric form can be substituted
for any L-amino acid residue, as long as the desired functional property
is retained by the polypeptide. NH2 refers to the free amino group
present at the amino terminus of a polypeptide. COOH refers to the free
carboxy group present at the carboxyl terminus of a polypeptide. In
keeping with standard polypeptide nomenclature described in J. Biol.
Chem., 243:3552-59 (1969) and adopted at 37 C.F.R.
§§1.821-1.822, abbreviations for amino acid residues are shown
in Table 2:

[0056]It should be noted that all amino acid residue sequences represented
herein by a formula have a left to right orientation in the conventional
direction of amino-terminus to carboxyl-terminus. In addition, the phrase
"amino acid residue" is defined to include the amino acids listed in the
Table of Correspondence and modified and unusual amino acids, such as
those referred to in 37 C.F.R. §§1.821-1.822, and incorporated
herein by reference. Furthermore, it should be noted that a dash at the
beginning or end of an amino acid residue sequence indicates a peptide
bond to a further sequence of one or more amino acid residues or to an
amino-terminal group such as NH2 or to a carboxyl-terminal group
such as COOH.

[0057]Guidance in determining which amino acid residues can be
substituted, inserted, or deleted without abolishing biological or
immunological activity can be found using computer programs well known in
the art, such as DNASTAR software. Preferably, amino acid changes in the
protein variants disclosed herein are conservative amino acid changes,
i.e., substitutions of similarly charged or uncharged amino acids. A
conservative amino acid change involves substitution of one of a family
of amino acids which are related in their side chains. Naturally
occurring amino acids are generally divided into four families: acidic
(aspartate, glutamate), basic (lysine, arginine, histidine), non-polar
(alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), and uncharged polar (glycine, asparagine,
glutamine, cystine, serine, threonine, tyrosine) amino acids.
Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly
as aromatic amino acids.

[0058]It is reasonable to expect that an isolated replacement of a leucine
with an isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar replacement of an amino acid with a
structurally related amino acid will not have a major effect on the
biological properties of the resulting variant.

[0059]Variants of the repressors and activators described herein include
glycosylated forms, aggregative conjugates with other molecules, and
covalent conjugates with unrelated chemical moieties (e.g., pegylated
molecules). Covalent variants can be prepared by linking functionalities
to groups which are found in the amino acid chain or at the N- or
C-terminal residue, as is known in the art. Variants also include allelic
variants, species variants, and muteins. Truncations or deletions of
regions which do not affect functional activity of the proteins are also
variants.

[0060]A subset of mutants, called muteins, is a group of polypeptides in
which neutral amino acids, such as serines, are substituted for cysteine
residues which do not participate in disulfide bonds. These mutants may
be stable over a broader temperature range than native secreted proteins
(see, e.g., Mark et al., U.S. Pat. No. 4,959,314).

[0062]It is reasonable to expect that an isolated replacement of a leucine
with an isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar replacement of an amino acid with a
structurally related amino acid will not have a major effect on the
biological properties of the resulting secreted protein or polypeptide
variant.

[0063]Truncations or deletions of regions which do not affect functional
activity of the proteins are also variants of the repressors and
activators described herein. Covalent variants can be prepared by linking
functionalities to groups which are found in the amino acid chain or at
the N- or C-terminal residue, as is known in the art.

[0064]It will be recognized in the art that some amino acid sequences of
the repressors and activators described herein of the invention can be
varied without significant effect on the structure or function of the
protein. If such differences in sequence are contemplated, it should be
remembered that there are critical areas on the protein which determine
activity. In general, it is possible to replace residues that form the
tertiary structure, provided that residues performing a similar function
are used. In other instances, the type of residue may be completely
unimportant if the alteration occurs at a non-critical region of the
protein. The replacement of amino acids can also change the selectivity
of binding to cell surface receptors (Ostade et al., Nature 361:266-268,
1993). Thus, the repressors and activators described herein of the
present invention may include one or more amino acid substitutions,
deletions or additions, either from natural mutations or human
manipulation.

[0065]Of particular interest are substitutions of charged amino acids with
another charged amino acid and with neutral or negatively charged amino
acids. The latter results in proteins with reduced positive charge to
improve the characteristics of the disclosed protein. The prevention of
aggregation is highly desirable. Aggregation of proteins not only results
in a loss of activity but can also be problematic when preparing
pharmaceutical formulations, because they can be immunogenic (see, e.g.,
Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al.,
Diabetes 36:838-845 (1987); and Cleland et al., Crit. Rev. Therapeutic
Drug Carrier Systems 10:307-377 (1993)).

[0066]Amino acids in the repressors and activators described herein of the
present invention that are essential for function can be identified by
methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085
(1989)). The latter procedure introduces single alanine mutations at
every residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as binding to a natural or synthetic
binding partner. Sites that are critical for ligand-receptor binding can
also be determined by structural analysis such as crystallization,
nuclear magnetic resonance or photoaffinity labeling (Smith et al., J.
Mol. Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312
(1992)).

[0067]As indicated, changes are preferably of a minor nature, such as
conservative amino acid substitutions that do not significantly affect
the folding or activity of the protein. Of course, the number of amino
acid substitutions a skilled artisan would make depends on many factors,
including those described above. Generally speaking, the number of
substitutions for any given repressor and activator as described herein
will not be more than 50, 40, 30, 25, 20, 15, 10, 5 or 3.

[0068]In addition, pegylation of the repressors and activators as
described herein and/or muteins is expected to provide such improved
properties as increased half-life, solubility, and protease resistance.
Pegylation is well known in the art.

Fusion Proteins

[0069]Fusion proteins comprising proteins or polypeptide fragments of the
repressors and activators as described herein can also be constructed.
Fusion proteins are useful for generating antibodies against amino acid
sequences and for use in various targeting and assay systems. For
example, fusion proteins can be used to increase the repressors and
activators described herein of the invention interaction with DNA or
which alter their biological function. Fusion proteins comprising a
signal sequence can be used.

[0070]A fusion protein comprises two protein segments fused together by
means of a peptide bond. Amino acid sequences for use in fusion proteins
of the invention can be utilize the amino acid sequences of the
repressors and activators as disclosed herein or can be prepared from
biologically active variants of those repressors and activators, such as
those described above. The first protein segment can include the
repressors and activators as described herein.

[0072]These fusions can be made, for example, by covalently linking two
protein segments or by standard procedures in the art of molecular
biology. Recombinant DNA methods can be used to prepare fusion proteins,
for example, by making a DNA construct which comprises a coding region
for the protein sequence of the repressors and activators as disclosed
herein in proper reading frame with a nucleotide encoding the second
protein segment and expressing the DNA construct in a host cell, as is
known in the art. Many kits for constructing fusion proteins are
available from companies that supply research labs with tools for
experiments, including, for example, Promega Corporation (Madison, Wis.),
Stratagene (La Jolla, Calif.), Clontech (Mountain View, Calif.), Santa
Cruz Biotechnology (Santa Cruz, Calif.), MBL International Corporation
(MIC; Watertown, Mass.), and Quantum Biotechnologies (Montreal, Canada;
1-888-DNA-KITS).

Example 1

Materials and Methods

[0073]Plasmid Construction

[0074]The pGL3O and pGL3T plasmids were generated by inserting DNA oligos
containing a lac operator and a tet operator flanked by a SacI and
HindIII sites (5'-3') into the pGL3 Basic (promega) vector using the
enzyme sites.

[0075]The luciferase reporter plasmid harboring the CMV promoter (pCMVL)
was constructed by PCR amplifying the promoter using the forward and
reverse primers harboring a NotI and AvrII site respectively. The
amplicon was digested with the NotI and AvrII and ligated into the pGL3O
vector digested with the NotI and SpeI.

[0076]The pCMVOL and pCMVT were generated by PCR amplifying the CMV
promoter using the same primers used to generated pCMVL. The amplicon was
digested with the NotI and AvrII and ligated into the pGL3O vector
digested with the NotI and AvrII.

[0077]The pLacI was constructed by replacing the 5' end (36 bp) and
EcoRV-PvuII flanked 150-bp regions of the pSYNIacI (from Dr. Scrable)
with the same regions from the wild type lacI. This was done by PCR
amplifying the 5' region of wild type lacI using primers harboring a
BamHI (forward) and an XbaI (reverse) sites the 150-bp region using
primers containing EcoRI and PvuII sites and inserting the amplicons into
the pSYNIacI using the enzyme sites.

[0078]The pNFlacI was generated by inserting the lacI in reverse
orientation (Jie Wei). The pLacIY was made by replacing the third amino
acid (proline) of the lacI with a tyrosine. This was done by PCR using a
forward primer harboring a BamHI site and a coding sequence for tyrosine
and a reverse primer with an XbaI (reverse) site. The PCR amplicon was
inserted into the pLacI using the BamHI and XbaI sites in the amplicon
and the vector.

[0079]The pTetR and pTetRNLS were generated by PCR amplifying the tetR
from the pcDNA6 vector using a forward primer containing a BamHI site and
a reverse primer containing an MluI site with and without NLS. The
amplicons were inserted into the pLacI vector using the BamHI and MluI
sites, which replaces the lacI with tetR or tetRNLS.

[0080]The pLacIGY was generated by inserting a glycine residue into the
lacIY at a position between the first amino acid (methionine) and the
second amino acid (lysine). This was done by PCR amplifying the lacIY
using a forward primer harboring a coding sequence of a glycine and a
BamHI sites and a reverse primer containing an XbaI site. The PCR
amplicon was inserted into pLacIY using the sites.

[0081]All the nested deletion constructs and lac operator containing
constructs were generated using the same strategy described in Example 4.

[0082]The pDL-IN was generated by PCR amplifying the rabbit beta-globin
intron from the K14-BG-PL-Rev vector using a forward primer containing a
SacI site and a reverse primer with an MluI site. The amplicon was
inserted into pDL5 using the same sites.

[0083]The phUBCOOL and the pSV4000L was constructed by PCR amplifying the
promoters using the forward and reverse primers harboring a NotI and SpeI
site respectively and ligating it into the pGL300 using the enzyme sites.

[0084]Luciferase Reporter Assay

[0085]NIH3T3 cell line was cultured in DMEM with 10% fetal bovine serum
(FBS). The empty vector was used as control for transfections of all
reporter plasmids. Each experiment was done in at least three biological
replicates. The results shown are the means of triplicate points. The
Dual Luciferase Reporter Assay Kit (Promega) was used to determine
luciferase expression, as a measure for promoter activity. After applying
40 μl of passive lysis buffer, firefly and renilla luciferase
expression was measured using a TD-20/20 tube luminometer (Turner
BioSystems) or the automated luminometer. 30 μl of firefly substrate
was injected in each well. Light intensity was measured. Then 30 μl
Stop&Glow renilla substrate was added and luminescence was measured
again. The ratio of firefly over renilla luminescence intensity was
calculated and used as a measure for promoter activity.

[0088]According to certain aspects, to achieve the reversible control of a
target gene expression in mammalians, two art-recognized prokaryotic
binary transcriptional regulatory systems, lac and tet
operator/repressor, were adopted. The functionality and reversibility of
both systems for transgenes have been previously demonstrated in mice
(Kistner et al., 1996)(Cronin et al., 2001). The Applicants compared the
two systems in a luciferase reporter assay. For repression comparisons,
the Lac operator (lacO) or Tet operator (tetO) sequence was placed in
between the CMV promoter and the luciferase gene. Plasmids harboring
repressors are engineered identically except for the region encoding each
repressor, thereby allowing the comparison of their repression capacity
per gene copy, which is a relevant comparison for a transgenic approach
(FIG. 3, panel A). Five different repressors were subjected to the
comparison: non-functional lac repressor (NFLacI), wild type lac
repressor (LacI), lac repressor with tight binding mutation (LacIY), tet
repressor (TetR), and tet repressor with nuclear localization signal
(TetRNLS). NFlacI was constructed by inserting the lacI in reverse
orientation to make it non-functional. LacIY was generated by adopting a
tight binding mutation identified from E. coli that was shown to enhance
binding affinity of the repressor to the operator, as disclosed herein
(Kolkhof et al., 1992). The same nuclear localization signal used for the
lac repressors was engineered into the tet repressor to produce tetRNLS.
LacO or tetO insertion alone had a modest deleterious effect on the
promoter in the absence of repressors (FIG. 3, panel B). The reporters
and repressors were transiently introduced into the NIH3T3 cells in a 1:1
molar ratio. All of the repressors, when compared to NFlacI, showed
repression of the CMV promoter at the given conditions (FIG. 3, panel C).

[0089]According to additional aspects, to further analyze the lac and tet
repressor systems, the repressors are tested on two different reporters
separately and side by side in different orders (FIG. 4, panel A). LacIY
and tetRNLS were used for the further comparison. Both lac and tet
repressors repressed the CMV promoter much more effectively when the
corresponding operators were located immediately downstream of the
promoter (FIG. 4, panels B and C). However, the repression by tetRNLS was
almost completely abolished when the tet operator was located after the
lac operator, indicating that the position of the operator is critical
for repression by the tet repressor. This observation is consistent with
the reliance of the prokaryotic repression mechanism on steric hindrance
(Schmitz and Galas, 1979)(Schmitz, 1981). The position of the operator
has to be optimal for the repressors to efficiently physically interfere
with transcription machinery. However, the repression by lacIY was far
less dependent upon the position of the operator. One explanation might
be that the tetramerized lac repressor is still able to physically
obstruct the transcription machinery even when the operator is positioned
further downstream of the promoter. Another possible explanation is that
the binding affinity of lacIY is strong enough to block transcription
elongation.

Example 3

Construction of Symmetric and Consensus Sequence Operators

[0090]The consensus operator is a 29-bp sequence, which is almost a
palindrome, but asymmetric by 4 mis-pairs. A 30-bp perfectly symmetric
lac operator sequence (SEQ ID NO:3) has been synthesized and shown to
interact with lac repressor at least 10 times more strongly than the
consensus sequence (FIG. 9, panel A and (Simons et al., 1984)).
Applicants used this modified lac operator to the system to improve the
binding affinity between the operator and the repressor. The reporter
with a symmetric operator sequence was better repressed by both lacI and
lacIY than the one with a consensus operator in the given experimental
condition (FIG. 9, panel B). Applicants' also doubled the number of
operator sequences to improve repression. In certain embodiments, this
alteration improves the binding kinetics between the operator and the
repressor. Results from doubling the operator sequence show that
repression was improved about 50%, when compared to those sequences
harboring only one operator sequence (FIG. 9, panel C).

Example 4

Development of a Bidirectional Cloning Strategy

[0091]Promoter characterization and operator insertion procedures can be
highly time-consuming and labor-intensive due to a large cloning burden
related partially to an inability to predict the location and the number
of operator insertion sites for a given promoter. This makes it difficult
to design the two procedures coherent with regard to the use of
restriction sites for generating deletion mutants and operator sequences
for each insertion site. To overcome this Applicants' devised a novel
cloning method, the bidirectional cloning strategy, which allowed us to
generate all the deletion mutant constructs and to insert the operators
at different locations using only three restriction enzymes (FIG. 17).
This method greatly accelerated the time-consuming and labor-intensive
procedures by simplifying and synchronizing the two steps, and is
applicable to any other promoter.

[0092]More specifically, the bi-directional cloning strategy is
accomplished by cloning PCR amplicons into pGL3 Basic Vector using AvrII
and SpeI sites. All PCR amplicons were generated to harbor sites at the
5' and 3' ends, respectively. These PCR amplicons were then digested with
AvrII and SpeI and inserted into pGL3 Basic vector using AvrII and SpeI
sites, which resulted in pDL. (FIG. 17, panel A, left). AvrII and SpeI
sites were also inserted into pGL3 Basic Vector via a lac operator
containing these two sites, resulting in pGLO. (FIG. 17, panel A, right).
All PCR amplicons in pDL vector can be modified in both 5' and 3'
directions. For 5' extensions, pDL is digested with AvrII and BsaI and
ligated with a fragment digested with SpeI and BsaI that contains an
insert to be added. For 3' extension, pDL is digested with SpeI and BsaI
and the excised fragment ligated with a pGLO vector digested with AvrII
and BsaI. Ligation between AvrII and SpeI destroys both sites, in the
final construct, the outer AvrII and SpeI sites are still unique and can
be used for further construction. (FIG. 17, panel B). Both 5' and 3'
extensions can be repeated unlimitedly, resulting in multiple copies of a
given sequence or introducing an entirely new sequence. (FIG. 17, panel
C). This procedure can be applied to any promoter using a pair of
comparable restriction sites absent in the given promoter sequence and a
third restriction site unique in the backbone plasmid.

Example 5

Development of a Conditional Regulatory System of Dnmt1 Gene Expression
Based on lac Operator/Repressor System In Vitro

[0093]Experimental Design and Results. In particular aspects, a binary
transcriptional regulation system based on the E. coli lacO/lacI system
(14, 17, 18) is provided. In certain aspects, the development of a binary
transcriptional repression system comprises the following two parts: (1)
Modification of Dnmt1 promoter with lacO insertion; and (2) Modification
of lacI gene.

[0094]The detailed experimental steps are the followings:

5.1 Characterization of the Dnmt1 promoter.5.2 Test of the effects of lacO
insertion with and without lac repressor in a reporter system.5.3
Insertion of multiple copies of the lacO sequence into the endogenous
Dnmt1 promoter using gene targeting.5.4 Analysis of the effects of the
targeted lacO insertion on endogenous Dnmt1 in the presence and absence
of lac repressor in cell culture.

5.1 Characterization of the Dnmt1 Promoter.

[0095]In particular aspects, identification of the locations in Dnmt1
promoter where insertions of the lac operator sequences can be made
without damaging promoter function are determined. This requires an
extensive characterization of Dnmt1 promoter. In the mouse genome, there
are three Dnmt1 promoters two of which are alternative promoters used in
oocytes (exon 1o) and in pachytene spermatocytes (exon 1 p) (19, 20). The
somatic promoter is located in between these two other promoters as drawn
in FIG. 11. Particular aspects comprise modification of the somatic
promoter located upstream of Exon 1s since this is the promoter used in
most tissues to drive Dnmt1 expression (FIG. 12). In additional aspects,
the DNA fragment located immediately upstream of Exon1s is subcloned into
a luciferase vector and subsequently analyze for promoter activity in
both undifferentiated embryonic stem cells and in NIH/3T3 fibroblasts,
using transient luciferase reporter assays. In further aspects, nested
deletion constructs and internal deletion constructs are generated to
identify putative transcriptional elements, based on two computer based
information, transcriptional binding factor search and the sequence
homology with other two species (Human and Rodent).

[0096]Results. A 3,461-bp MspI fragment derived from a genomic phage
library constructed with 129/SvJae mouse genomic DNA was subcloned. This
MspI fragment extends from 3,418 by upstream of the transcription
initiation site to 99 by downstream of the transcription initiation site
(FIG. 12, panel A). The 3' end of this fragment is located 17 by upstream
of the translation initiation site. This fragment was cloned into a
luciferase reporter plasmid and tested the promoter activity in NIH/3T3
cells. This fragment showed about a half of promoter activity as compared
to SV40 promoter in our transient transfection assay. A series of 5', 3',
and internal deletion mutants of the promoter were generated, and those
mutants tested in reporter system. All of the deletion mutants were
designed such that each break point in the promoter does not reside
within either the putative transcription factor binding sites or the
regions with high homology amongst the three species so that those break
points can be used for operator insertion without breaking putative
cis-elements.

[0097]Increasing promoter activity was observed as the promoter was
deleted from the 5' end to -182 by upstream of the TSS, suggesting the
presence of repressor binding site(s) within the deleted regions (FIG.
12, panel A). On the other hand, as the promoter was further deleted from
-182 to -16 by upstream of the TSS, promoter activity gradually decreased
by several fold. This result suggests that the region (-182 to -16 bp)
contains several important cis-elements as reported previously (Kishikawa
et al., 2002)(Kimura et al., 2003). Some of the putative transcriptional
cis-elements have been further characterized. Results from the 3' and
internal deletion constructs confirmed the observations from the 5'
deletion mutants (FIG. 12, panel B).

5.2 Test of the Effects of lacO Insertion with and without Lac Repressor
in a Reporter System.

[0098]The effects of lacO insertion with and without lac repressor in a
reporter system are tested (FIG. 13, panel A). As stated above, a system
is desired that will allow regulation of Dnmt1 expression only in a
desired specific tissue and at a specific time. This comprises
identification of locations where lacO insertion has no effect on the
promoter activity. With identified lacO insertion sites that do not
affect expression in either of the two tested cell types, various
combinations of lacO insertion at multiple locations are tested to
determine the number of locations to insert lacO sequences to get the
best repression with transiently transfected lac repressor.

[0099]Based on Applicants reporter assay data with deletion mutants,
several locations for lacO insertion were chosen around the putative
transcriptional cis-elements. For lacO insertion, a bidirectional cloning
strategy (See Example 4) was developed that allowed inserting lacO
sequences at different locations using the same pair of compatible
restriction enzymes (SpeI and AvrII) without involving screening steps.
With this strategy, more than 200 constructs were generated with
different numbers of lacO sequences at different locations with a
homogeneous sequence at each junction in relatively short time. The
effect of lacO insertion at each individual site on the promoter activity
was tested with and without lac repressor, and three sites (-182, -59,
and +99) were identified that can accommodate lacO insertion with almost
no negative effect on promoter activity. The lacO sequences were then
inserted at multiple locations to determine the number of lacO sequences
to be inserted to get the best repression. Operators at the three
locations were enough to successfully repress Dnmt1 promoter in the
presence of lac repressor in the transient transfection reporter assay.
In addition, the symmetric operator and double-operator sequences were
applied to the three locations (-182, -59, and +99) in all possible
combinations (FIG. 13, panel B).

5.3 Insertion of Multiple Copies of the lacO Sequence into the Endogenous
Dnmt1 Promoter Using Gene Targeting.

[0100]Insertion of Multiple copies of the lacO sequence are inserted into
the endogenous Dnmt1 promoter using gene targeting. Applicants obtained
approximately 22 kb of genomic DNA subcloned from a phage library derived
from 129/SvJae genomic DNA, stretching from about 4 kb upstream of Exon1s
to 18 kb downstream of the transcription start site. A gene targeting
vector was constructed using these isogenic genomic fragments to ensure
efficient gene targeting (23). In certain aspects, the gene targeting is
perform on wild-type J1 ES cells. In certain aspects, the lacO sequence
is introduced into the endogenous Dnmt1 promoter, using a
replacement-type gene-targeting vector. After homologous recombination,
correctly targeted alleles contain selectable markers. In particular
aspects, this selectable marker is removed by Cre recombinase, leaving
one loxP site in intron 1 about 700 bp downstream from exon1s. In
principle, at least three possible consequences can result from having
this loxP in the intron 1. First, it can break a cis-element located at
the site where the loxP is inserted. Second, it can have an effect on
promoter activity by changing the distance between promoter and
cis-elements located downstream of the loxP site. Third, it can interfere
with splicing. Although the second and third possibilities can not be
ruled out, they are less likely to be a problem since the effect of 34 by
increase in distance on the activity of cis-elements located more than
700 by downstream from the promoter is unlikely to be substantial, and it
is also less likely that a cis-element involved in splicing would be
located at more than 500 by downstream of splicing donor site and at more
than 9 kb upstream of splicing acceptor site. A Web-based putative
transcription factor search was performed with the sequence around the
loxP site to check the first possibility. No putative transcription
factor binding sites were found with a score higher than ninety in the
sequence. Low CpG density in intron 1 reduces the possibility of the
presence of a strong enhancer element around the loxP site. An
alternative way that does not leave any exogenous sequence besides lacO
sequences in the targeted allele is to use a hit-and-run strategy, using
an insertion-type gene-targeting vector (24). This strategy requires two
round of homologous recombination, which extends ES cell culture, hence
making it more difficult to keep ES cell undifferentiated.

5.4 Analysis of the effects of the targeted lacO insertion on endogenous
Dnmt1 in the presence and absence of lac repressor in cell culture.

[0101]Analysis of the effects of the targeted lacO insertion in the
presence and absence of lac repressor in cell culture. Upon successfully
introducing lacO sequences into one allele of the endogenous Dnmt1 using
gene targeting, a lacI gene is stably introduced into this heterozygously
targeted ES cells, and a quantitative real-time RT-PCR is performed to
measure the expression level of Dnmt1 in this one allele targeted ES
cells. A quantitative real-time RT-PCR is specifically designed to
differentiate the targeted allele from the wild type allele by taking
advantage of the fact that the targeted allele has two lacO sequences in
5'UTR region. Preferably, the expression level targeted to achieve from
the targeted allele is 10% of that of wild type allele. In additional
aspects, the allele is introduced into the mouse germline, to generate
lacI transgenics. A couple of modifications have been made in lacI gene
to enhance its repressor function (described in experimental design and
Example 6).

Example 6

Modification of the Lac Repressor

[0102]The degree of transcriptional repression by operator/repressor based
prokaryotic binary systems is dependant on multiple factors, including
the binding affinity between the operator and the repressor, the amount
of repressor present in the biological system, the strength of the
transcription unit against which the binary repression system competes,
and the position of the operator (Scrable, 2002). Of these factors,
binding affinity and the amount of repressor are the factors which
determine the repression capacity of the system. Therefore, enhancing
binding affinity and increasing the quantity of repressor would increase
the reliability and general applicability of a binary system. Applicants
have modified the lacI gene to 1) stabilize the Lac repressor, which
results in increasing the quantity of the repressor and 2) increase the
Lac repressors affinity for the Lac operator.

6.1 Quantitative Improvement of the lac System by Stabilizing Amino Acid
Insertion

[0103]The Applicants modified the repressor to include a stabilizing amino
acid insertion based on the N-end rule (Varshaysky, 1996) to increase the
repressor protein quantity by increasing the half-life of the protein.
The N-terminal amino acid sequence of lacI protein is lysine, which is
known to destabilize proteins by being a target site for ubiquitination
when located at the N-terminus of a protein. Among exemplary stabilizing
amino acids, three amino acids were initially selected based on the
following two criteria: 1) amino acid encoded by a codon with high codon
usage and 2) amino acid encoded by a codon starting with guanine. The
first criterion is for better translation efficiency, and the second
criterion is to increase the rate of translation initiation by completing
the consensus eukaryotic translation initiation sequence. Glycine and
valine were chosen because these two amino acids meet both criteria.
Alanine was also chosen since it meets both criteria, although it is not
a stabilizing amino acid in mammal.

[0104]Applicants introduced a glycine, a valine, or an alanine, a known
stabilizing amino acid, to the N-terminus of lacIY before the lysine
(lacIAY and IacIGY). This insertion not only adds a stabilizing amino
acid to the repressor, but also completes the Kozak sequence, the
consensus eukaryotic translation initiation sequence (GCCACCATGG) (SEQ ID
NO:4). In wild type lacI, the guanine after the ATG, which is known to
play an important role in translation initiation (Kozak, 1997), is
missing because next codon encoding lysine starts with an adenine (AAA).
Applicants restored this critical guanine residue by insertion of a
glycine (GGC) or alanine (GCC). Completion of the Kozak sequence could
potentially increase the quantity of repressor protein through improved
translation efficiency.

[0105]To test whether the insertion of the stabilizing amino acid indeed
increased the amount of repressor and more importantly resulted in an
improved repression capacity, the Applicants compared the repression
capabilities and stabilities of lac repressors with and without the
stabilizing amino acid. To compare the repressors for their repression
capabilities, the Applicants performed reporter assays with different
conditions. A fixed amount (1 mg) of a reporter plasmid was
co-transfected with increased amounts of each repressor (50 ng, 200 ng, 1
mg, and 2 mg) (FIG. 7, panel A). Both lacIAY and lacIGY repressed the CMV
promoter substantially better than the repressors without the stabilizing
amino acid at all four conditions, and lacIGY showed the best repression
(FIG. 7, panel A). This result indicates that the insertion of the
stabilizing amino acid increased the repression capacity of the lac
repressor. To test the effect of the modification on the stability of lac
repressor, we co-transfected the lac repressor containing plasmids with a
plasmid carrying Flag-tagged Dnmt3b4, which serves as a normalizing
control, and measured protein quantity of each repressor at different
time points after the transfection by western blot. Although the same
amount of each plasmid was transfected, more repressor protein was
detected for lacIGY than lacIY over time while the Dnmt3b4 protein
remained constant (FIG. 7, panel B). This suggests that the insertion of
the glycine resulted in increased protein levels through increased
stability of the protein rather than improved translational efficiency.

[0106]Results. Lac repressor modified with those three amino acids were
constructed, and tested in reporter assay system. All lac repressor with
this modification showed increased repression ability. The best result
was observed with lac repressor with glycine addition (˜2 times
better than wild type repressor).

6.2 Engineering lacI Gene with Tight Binding Mutation

[0107]Two tight binding mutations (P3Y and S61 L) have been introduced
into lacI gene, and showed tighter binding affinity to its operator
sequence in Ecoli (25). In particular aspects, these tight binding
mutants are introduced into mammalian cells (e.g., to provide enhanced
repression ability in luciferase reporter assay system in NIH/3T3 cell
line).

[0108]Results. Three lac repressors with tight binding mutation (P3Y, S61
L, and P3Y&S61 L) have been constructed and tested these mutants in the
Applicants' reporter assay system with luciferase gene driven by Dnmt1
promoter modified with lacO sequences in NIH/3T3 cell line. Of the three
tight binding mutants, P3Y mutants showed the best repression ability,
˜5 times better than wild type repressor similar to the results
seen in Ecoli (25).

6.3 Generation of lac Activator by Fusing lac Repressor with VP16

[0109]Generation of lac activator by fusing lac repressor with VP16. With
access to the Dnmt1 promoter through lac operator/repressor interaction,
this interaction is, according to particular aspects used to overexpress
Dnmt1 using lac repressor fused with transcriptional activator, VP16. Lac
repressor/VP16 fusion protein has been successfully used in mammalian
cells (27). With overexpression of Dnmt1 by lac repressor/VP16, the
inventive binary transcriptional regulation system allows to not only
down-regulate, but also up-regulate endogenous Dnmt1 expression in vivo.
Additional aspects provide various Dnmt1 promoters with lacO at different
locations to provide optimal locations for activation using luciferase
reporter assay.

[0110]VP16 was introduced to lacI gene at three locations, one of which is
the same location described in (27). Two other locations were chosen
based on the knowledge of functional domains of lac repressor and
restriction sites availability. Both tight binding mutant P3Y and wild
type lac repressor were engineered. Tight binding mutants with VP16
fusion showed better activation ability than wild type lac
repressor/VP16. Dnmt1 promoter was overexpressed up to 2.5-fold with one
of tight binding mutant/VP16 constructs (LAVPN) in transient transfection
reporter assay in NIH/3T3 cells. By introducing multiple VP16s (3 and 4
in a row), Dnmt1 was overexpressed up to 4-fold.

Example 7

Reversible Repression of the Dnmt1 Promoter by the lac Repressor and IPTG
was Demonstrated

[0111]Applicants tested whether the lac repressor can repress the modified
Dnmt1 promoter. A reporter plasmid with the modified Dnmt1 promoter and
the lacI or lacIGY plasmids were transiently introduced into NIH3T3 cells
in a 1:1 molar ratio. Both lac repressors successfully repressed the
operator inserted Dnmt1 promoter (FIG. 10, panel A). Almost complete
repression (3.7% residual expression) was achieved with lacIGY. The
repression by lacI was completely reversed by 0.5 mM IPTG treatment while
only 25% restoration was achieved for lacIGY with the same IPTG
treatment. To test whether the repression by lacIGY can be completely
reversed by higher amounts of IPTG, increased molar concentrations of
IPTG (0.5 mM to 10 mM) were applied (FIG. 10, panel B). The best
restoration levels of transcriptional expression was at 3 mM IPTG, which
was 40% of unregulated expression.

Example 8

General Applicability of the lac System was Demonstrated

[0112]To test the general applicability of the repression principle
Applicants applied the lac system to other promoters. Applicants modified
the simian virus 40 (SV40) and the human ubiquitin C (hUbc) promoters
with lac operators and subjected them to a reporter assay. As seen with
the CMV and the Dnmt1 promoters, these promoters were almost completely
repressed by the modified lac repressor (FIG. 14, panel A). According to
certain embodiments, this result indicates that the modifications the
Applicants introduced to the lac operator/repressor system have
substantially improved reliability and general applicability of the
system and suggests that our repression approach can be successfully
applied to other eukaryotic promoters.

Example 9

Blocking Transcription Elongation by lac Repressor was Demonstrated

[0113]According to certain embodiments, the best placement of the lac
operator is within intronic regions. Reasons for this example include: 1)
a significant portion of eukaryotic genes has more than one promoter, 2)
insertion locations identified with a certain cell type may not be
optimal for other cell types, and 3) some genes may not be able to
accommodate operator sequences at their 5'UTR, the most crucial location
for the repression. In certain embodiments the issues listed above may be
addressed by inserting the lac operator sequences into intronic regions
if the repressor is able to impede transcription elongation. In certain
embodiments, the modifications that Applicants integrated into the lac
operator/repressor system might allow the blockage of transcription
elongation through the enhanced operator-repressor interaction. To test
this, the rabbit β-globin intron was modified with the symmetric
double operators under the control of Dnmt1 promoter. In a transient
reporter assay, expression from the Dnmt1 promoter was significantly
repressed, indicating that the modified lac repressor (lacIGY) was able
to block the transcription elongation (FIG. 14, panel B). According to
certain embodiments, the relatively close location of the lac operators
to the promoter (448 by downstream from the transcription start site)
allowed the lac repressors to interfere with transcription initiation.

Example 10

A Conditional Regulatory System of Dnmt1 Gene Expression in the Mouse is
Developed

[0114]Introduction of the Dnmt1-lacO allele into the mouse germline. In
particular aspects, the targeted allele is introduced into the mouse
germline. In certain embodiments, the targeted allele is maintained in
129/svJae mice, and is optionally also backcrossed to C57BL/6 for testing
with lacI transgenics.

[0115]Generation of lacI transgenic mice and testing of the binary system.
In certain aspects, two lines of transgenic mice are generated, one with
ubiquitous lacI expression and another with tissue specific lacI
expression to test our binary system at the mouse and a tissue level.
(28) For the former, three promoters, ubiquitine, EF1α, and human
β-actin promoters are first compared. The strongest promoter to have
a robust ubiquitous lac repressor or activator expression is chosen. For
the latter, the FabpI4x at-132 modified fatty acid binding promoter
(28) is preferably used, which is well-characterized and provides
excellent tissue-specific expression in the colonic crypt epithelium and
small intestine, with some additional expression in the bladder (29).
Quantitative RT-PCR is performed to confirm that this binary system works
in Dnmt1-lacO mice carrying the lacI transgene, compared to
non-transgenic sibling Dnmt1-lacO controls. In particular aspects of this
binary transcriptional repression/activation system transcriptional
repression/activation can be reversed by IPTG (17). Transgenic mice are
first generated expressing lac repressor/activator with tight binding
mutation and stabilizing amino acid. Alternatively, lacI gene without
these modifications can be used. In additional aspects, the system is
expanded to include tissue-specific expression in other tissue types for
modulating and determining the effects of modulated Dnmt1 expression on
tumor model systems.

Example 11

A Conditional Regulatory System of Dnmt1 Gene Expression in the Mouse was
Demonstrated

[0116]This example describes the in vivo demonstration of the repression
system. After the establishment of the binary systems in vitro,
Applicants tested the system in mice. Applicants introduced the lac
operators to the endogenous Dnmt1 promoter through gene-targeting, and
generated transgenic lines expressing the lac repressor. In the mouse
experiments, Applicants found that the improved lac system successfully
repressed endogenous Dnmt1 expression tissue-specifically and
ubiquitously. The repression was potent enough to reproduce the embryonic
lethal phenotype of genetic knock-outs when applied ubiquitously.
Importantly, this lethal phenotype was rescued by treating the pregnant
mice with IPTG.

[0117]Due to the essential requirement of Dnmt1 for development and
viability, in vivo characterization of Dnmt1 functions through knock-out
approaches have been very limited (Li et al., 1992; Jackson-Grusby et
al., 2001; Lei et al., 1996; Holm et al., 2005). Now, with this novel
approach, Applicants circumvented the lethality problem and successfully
produced Dnmt1 knockdown mouse models for the first time, which could
serve as a useful resource for studying the role of DNA methylation in
many biological processes and diseases.

Example 12

Induced Overexpression of Endogenous Dnmt1 was Demonstrated

[0118]Applicants applied the binary activation system to the endogenous
Dnmt1 to test the novel gene activation principle on an endogenous gene.
To induce overexpression of Dnmt1 in mice, Applicants generated Dnmt1LGT
mice that contain tet operators in the endogenous Dnmt1 promoter in
addition to the lac operaor sequences. Mice were then crossed with
existing tetVP16 or rtetVP16 transgenic lines. No successful transgenic
mouse models have been reported for Dnmt1 upregulation. However, the
approach enabled production of mouse models exhibiting elevated Dnmt1
expression in various tissues. These mouse models provide a new
opportunity for the functional analysis of Dnmt1 and facilitate the
gain-of-function genetics for Dnmt1.

Example 13

The Role of DNA Methylation in Cancer Using Binary Transcriptional
Regulatory System in Mouse Models

[0119]With the desired level of Dnmt1 expression (repression and
activation), which does not cause non-viability of either cells or mice,
the role of DNA methylation in cancer is investigated with this system in
following three mouse experiments: [0120]1) Downregulation of Dnmt1 in
mice predisposed to develop cancer [0121]2) Overexpression of Dnmt1 in
normal mice and in the mice predisposed to develop cancer [0122]3)
Downregulation of Dnmt1 in the mice predisposed to develop cancer only
after tumor formation. [0123]For the first experiment, a Dnmt1-lacO mouse
carrying lacI transgene is crossed with ApcMin/+ mice and
Mlh.sup.-/- mice. With Dnmt1 heterozygous and hypomorphic mice
(Dnmt1.sup.-/R), we have previously shown that reduced expression of
Dnmt1 results in the suppression of benign neoplasia in the intestines of
ApcMin/+ mice and of benign and malignant intestinal tumors in
Mlh.sup.-/- mice (30, 31). In particular aspects, Dnmt1 expression is
repressed as low as that of Dnmt1.sup.-/R mice. The possible effects of
lacI transgene on tumorigenesis are controlled by crossing lacI
transgenic mice with ApcMin/+ mice and Mlh.sup.-/- mice. The
difference in the underlying mechanisms for the reduced expression of
Dmnt1 between Dnmt1.sup.-/R and Dnmt1-lacO mouse carrying lad transgene
may result in different phenotypes. The repression mechanism of
Dnmt1.sup.-/R mice does not involve trans-acting factors, but that of
Dnmt1-lacO mouse carrying lacI transgene involves trans-acting factor,
lac repressor. It is possible but not necessary that reduced expression
level of Dnmt1 in Dnmt1-lacO mouse carrying lacI transgene might be not
as stable as that of Dnmt1.sup.-/R over the time of mouse development.
For the second experiment, a Dnmt1-lacO mouse carrying lac/VP16 transgene
is crossed with normal mice, ApcMin/+ mice and Mlh.sup.-/- mice to
confirm that Dnmt1 overexpression enhances tumorigenesis. Where no
phenotypic changes are observed, several interpretations are possible.
First, together with the results from the experiment with Dnmt1.sup.-/R
and ApcMin/+ mice it suggests that sufficient Dnmt1 expression level
is required for and involved in tumorigenesis, but Dnmt1 dose not play a
causal role. Second, Dnmt1 may have a causal effect, but Overexpression
of Dnmt1 alone is not sufficient to enhance tumorigenesis. Third Dnmt1
may play a causal role in tumorigenesis, but not through its
overexpression. For third experiment, the same cross is performed as for
the first experiment. But, in this experiment, lac repressor is blocked
by IPTG. It has been shown that IPTG could act transplacentally in the
mouse (17), so if the mother is treated with IPTG (10 mM IPTG in drinking
water), lac repressor can be blocked in the offspring, resulting normal
level of Dnmt1 expression. IPTG treatment will be stopped after the
offspring develop tumor to confirm the role of DNA methylation in tumor
progression and maintenance. ApcMin/+ mice and Mlh.sup.-/- mice
treated with IPTG are included in certain aspects of this experiment as a
control for the effect of IPTG on tumorigenesis.